Physicochemical Profiling of α‐Lipoic Acid and Related Compounds

Lipoic acid, the biomolecule of vital importance following glycolysis, shows diversity in its thiol/disulfide equilibria and also in its eight different protonation forms of the reduced molecule. In this paper, lipoic acid, lipoamide, and their dihydro derivatives were studied to quantify their solubility, acid–base, and lipophilicity properties at a submolecular level. The acid–base properties are characterized in terms of six macroscopic, 12 microscopic protonation constants, and three interactivity parameters. The species‐specific basicities, the pH‐dependent distribution of the microspecies, and lipophilicity parameters are interpreted by various intramolecular effects, and contribute to understanding the antioxidant, chelate‐forming, and enzyme cofactor behavior of the molecules observed.     

In silico Prediction of Human Oral Absorption Based on QSAR Analyses of PAMPA Permeability

The parallel artificial membrane permeation assay (PAMPA) was developed as a model for the prediction of transcellular permeation in the process of drug absorption. Our research group has measured the PAMPA permeability of peptide‐related compounds, diverse drugs, and agrochemicals. This work led to a classical quantitative structure–activity relationship (QSAR) equation for PAMPA permeability coefficients of structurally diverse compounds based on simple physicochemical parameters such as lipophilicity at a particular pH (log P_oct and |pK_a?pH|), H‐bond acceptor ability (SA_HA), and H‐bond donor ability (SA_HD). Since the PAMPA permeability of lipophilic compounds decreased with their apparent lipophilicity due to the unstirred water layer (UWL) barrier on membrane surfaces and to membrane retention, a bilinear QSAR model was introduced to explain the permeability of a broader set of compounds using the same physicochemical parameters as those used for the linear model. We also compared PAMPA and Caco‐2 cell permeability coefficients of compounds transported by various absorption mechanisms. The compounds were classified according to their absorption pathway (passively transported compounds, actively transported compounds, and compounds excreted by efflux systems) in the plot of Caco‐2 vs. PAMPA permeability. Finally, based on the QSAR analyses of PAMPA permeability, an in silico prediction model of human oral absorption for possibly transported compounds was proposed, and the usefulness of the model was examined.     

Interfacial ionization and partitioning of membrane-bound local anesthetics.

Consideration of the interfacial protonation equilibria of membrane-associated amphiphiles indicates that the partition coefficients of the protonated and unprotonated species will differ considerably. The partition coefficients of the charged and uncharged forms of spin-labelled myristic acid in dimyristoylphosphatidylcholine bilayer dispersions have been measured by EPR spectroscopy and found to be approximately 140-fold higher for the protonated acid than for the dissociated salt form. This ratio of partition coefficients is found to be in good agreement with that predicted from the interfacial shift in pKa of the fatty acid on its partitioning into the membrane. The latter was determined from the changes in the EPR spectra of the membrane-associated fatty acid with pH and was found to be +2.1 pH units. The interfacial shifts in pKa for a series of spin-labelled analogues of tertiary amine local anaesthetics have been determined from the pH dependence of the partition coefficients in dimyristoylphosphatidylcholine bilayer dispersions and are found mostly to be in the range of approx. -1.0 to -1.5 pH units, corresponding to a 10- to 30-fold higher partition coefficient of the uncharged base compared with that of the charged ammonium form.     

Derivative spectrophotometry as a tool for the determination of drug partition coefficients in water/dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) liposomes.

The partition coefficients (K(p)) between lipid bilayers of dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) unilamellar liposomes and water were determined using derivative spectrophotometry for chlordiazepoxide (benzodiazepine), isoniazid and rifampicin (tuberculostatic drugs) and dibucaine (local anaesthetic). A comparison of the K(p) values in water/DMPG with those in water/DMPC (dimyristoyl-L-alpha-phosphatidylcholine) revealed that for chlordiazepoxide and isoniazid, neutral drugs at physiological pH, the partition coefficients are similar in anionic (DMPG) and zwitterionic (DMPC) liposomes. However, for ionised drugs at physiological pH, the electrostatic interactions are different with DMPG and DMPC, with the cationic dibucaine having a stronger interaction with DMPG, and the anionic rifampicin having a much larger K(p) in zwitterionic DMPC. These results show that liposomes are a better model membrane than an isotropic two-phase solvent system, such as water-octanol, to predict drug-membrane partition coefficients, as they mimic better the hydrophobic part and the outer polar charged surface of the phospholipids of natural membranes.     

Cytostatic/Cytotoxic Effects of 5‐Fluorouridine Nucleolipids on Colon,Hepatocellular, and Renal Carcinoma Cells: in vitro Identification of a Potential Cytotoxic Multi‐Anticancer Drug

The insufficient penetration through the cell membranes is one of the major drawbacks of chemotherapeutics such as 5‐fluorouracil (5‐FU; 1 ). To improve the penetration, a useful strategy is the attachment of lipophilic moieties. Thus, we have synthesized a series of nucleolipid derivatives of 5‐fluorouridine (5‐FUrd; 2a ), carrying lipophilic moieties at N(3) and/or at the 2′,3′‐O position, i.e., 3a, 3b, 4 – 7 , and tested their cytostatic/cytotoxic activities towards three carcinoma cell lines (colon (HT‐29), hepatocellular (HepG2), and renal (RENCA)) in comparison with 5‐FU ( 1 ) and 5‐FUrd ( 2a ). After 48 h of incubation, four derivatives, 3a, 3b, 5 , and 7 , showed inhibitory effects on the survival of HT‐29, HepG2, and RENCA cells. Additionally, to differentiate between anticancer and side‐effects, we tested the cytotoxicity of the derivatives in human macrophages. Interestingly, the derivatives 4, 5 , and 6 did not exhibit any effects on survival of THP‐1 macrophages. Furthermore, we investigated the apoptosis induction of compound 1 and 2a , and the above‐mentioned derivatives in HT‐29 cells. Derivative 5 showed the highest significant (p<0.05; p<0.01) increase of the apoptosis at 80 μM after 2‐h or 4‐h treatment, as well as after 6‐h incubation at 40 μM (p<0.05). Real‐time PCR revealed that 40‐μM derivative 5 showed a 1.8‐fold increase of the pro‐apoptotic caspase‐3 gene and a twofold significant increase (p<0.01 and p<0.05 vs. control and 1 , resp.) of the tumor suppressor TP53 gene, whereas the other compounds did not show any effect. We demonstrated that some 5‐FUrd derivatives such as compound 5 are more effective than 5‐FU or 5‐FUrd concerning a cytotoxic (vs. cytostatic (5‐FU, 5‐FUrd)) effect on different cancer cell lines, but without cytotoxic side‐effects on differentiated macrophages. Thus, compound 5 is suggested as a novel potent cytotoxic multi‐anti‐cancer drug.     

Site-specific basicities regulate molecular recognition in receptor binding: in silico docking of thyroid hormones

Interactions between thyroid hormone α and β receptors and the eight protonation microspecies of each of the main thyroid hormones (thyroxine, liothyronine, and reverse liothyronine) were investigated and quantitated by molecular modeling. Flexible docking of the various protonation forms of thyroid hormones and high-affinity thyromimetics to the two thyroid receptors was carried out. In this method the role of the ionization state of each basic site could be studied in the composite process of molecular recognition. Our results quantitate at the molecular level how the ionization state and the charge distribution influence the protein binding. The anionic form of the carboxyl group (i.e., carboxylate site) is essential for protein binding, whereas the protonated form of amino group worsens the binding. The protonation state of the phenolate plays a less important role in the receptor affinity; its protonation, however, alters the electron density and the concomitant stacking propensity of the aromatic rings, resulting in a different binding score. The combined results of docking and microspeciation studies show that microspecies with the highest concentration at the pH of blood are not the strongest binding ones. The calculated binding free energy values can be well interpreted in terms of the interactions between the actual sites of the microspecies and the receptor amino acids. Our docking results were validated and compared with biological data from the literature. Since the thyroid hormone receptors influence several physiologic functions, such as metabolic rate, cholesterol and triglyceride levels, and heart frequency, our binding results provide a molecular basis for drug design and development in related therapeutic indications.     

Partition coefficients (n-octanol/water) of N-butyl-p-aminobenzoate and other local anesthetics measured by reversed-phase high-performance liquid chromatography

For the determination of the logarithmic partition coefficients between n-octanol and water (log P_o/w) of local anesthetics, the pH of the aqueous phase needs to be adjusted to high values to ensure that the local anesthetics are in the unionized form. Using the shake-flask or the stir-flask method, this high pH may catalyze hydrolysis, leading to increasing amounts of impurities in time. These impurities exclude non-selective quantification methods like UV spectrometry and require repetitive quantitative analysis of both liquid phases resulting in a tedious and time-consuming method. A rapid reversed-phase HPLC method was developed to measure log P_o/w of the local anesthetics N-butyl-p-aminobenzoate, methyl-p-aminobenzoate, benzocaine, procaine, mepivacaine, prilocaine, lidocaine, bupivacaine, etidocaine, tetracaine and oxubuprocaine.     

Effect of lipophilicity of Mn (III) ortho N-alkylpyridyl- and diortho N,N′-diethylimidazolylporphyrins in two in-vitro models of oxygen and glucose deprivation-induced neuronal death

In vivo investigations have confirmed the beneficial effects of hydrophilic, cationic Mn(III) porphyrin-based catalytic antioxidants in different models of oxidative stress. Using a cell culture model of rat mixed neuronal/glial cells, this study investigated the effect of MnTnOct-2-PyP⁵⁺ on oxygen and glucose deprivation (OGD)-induced cell death as compared to the effects of widely studied hydrophilic analogues MnTE-2-PyP⁵⁺ and MnTDE-2-ImP⁵⁺ and a standard compound, dizocilpine (MK-801). It was hypothesized that the octylpyridylporphyrin, MnTnOct-2-PyP⁵⁺, a lipophilic but equally potent antioxidant as the other two porphyrins, would be more efficacious in reducing OGD-induced cell death due to its higher bioavailability. Cell death was evaluated at 24 h using lactate dehydrogenase (LDH) release and propidium iodide staining. At concentrations from 3–100 µm, all three porphyrins reduced cell death as compared to cultures exposed to OGD alone, the effects depending upon the concentrations and type of treatment. To assess the effect of lipophilicity the additional experiments were performed using submicromolar concentrations of MnTnOct-2-PyP⁵⁺ in an organotypic hippocampal slice model of OGD with propidium iodide and Sytox staining. When compared to oxygen and glucose deprivation alone, concentrations of MnTnOct-2-PyP⁵⁺ as low as 0.01 µm significantly (p<0.001; power 1.0) reduced neuronal cells similar to control. This is the first in vitro study on the mammalian cells which indicates that MnTnOct-2-PyP⁵⁺ is up to 3000-fold more efficacious than equally potent hydrophilic analogues, due entirely to its increased bioavailability. Such remarkable increase in efficacy parallels 5.7-orders of magnitude increase in lipophilicity of MnTnOct-2-PyP⁵⁺ (log P=?0.77) when compared to MnTE-2-PyP⁵⁺ (log P_ow=?6.43), P_ow being partition coefficient between n-octanol and water.     

Interaction of molecular probes with living cells and tissues. Part 2

Summary Cultured rat fibroblasts were exposed to 41 cationic fluorescent probes of very varied hydrophilicity/lipophilicity. Outcome of probe-cell interaction fell into one of the following categories: probe failed to enter the cells; probe accumulated on cell surfaces; probe accumulated in mitochondria, and/or in other intracellular regions. The observations were analysed using a Simplistic Chinese Box (SCB) approach, and the following conclusions were reached. It was the hydrophilic probes which failed to enter cells, whilst extremely lipophilic probes were retained on the cell surfaces. Only the slightly lipophilic cationic probes were permeant, and accumulated in mitochondria. Using the probes log P values to model hydrophilicity/lipophilicity, effective cationic mitochondrial stains can be specified numerically so: 0P _probe<+5. This SCB model was used to rationalise a variety of earlier observations on the action of mitochondrial probes. The applicability of the SCB approach to integrate image-based and biochemical investigations was demonstrated by using the action of chlorpromazine on mitochondrial action as a case example.     

Membrane incorporation and induction of secondary structure of synthetic peptides corresponding to the N-terminal signal sequences of the glucitol and mannitol permeases of Escherichia coli

The 22-residue synthetic signal peptide of the glucitol permease (Enzyme IIgut of the bacterial phosphotransferase system; gut22), which in the intact protein is believed to function in envelope targeting, was found to insert into phospholipid monolayers of various phospholipid compositions up to high limiting pressures (36-41 milliNewton/m). The partition coefficient, derived from monolayer area expansion experiments, was greatest for the negatively charged gut22 when partitioning into monolayers of the zwitterionic lipid 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (about 1.1 X 10(5] as compared with that obtained with a mixture of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine and the negatively charged lipids 1-palmitoyl-2-oleoyl-3-sn-phosphatidylglycerol and cardiolipin. Gut22 contains a titratable histidyl residue (pKa = 6.8), and its protonation decreased the relative monolayer area increase 3-fold. Circular dichroism spectra showed that gut22 formed an amphiphilic alpha-helix when incorporated into lipid membranes (estimated percent helix = 65%). Fluorescence measurements indicated that tryptophan 11 is in a more hydrophobic environment in the presence of lipid than in its absence, with the environment being more hydrophobic at pH 5 than at pH 8. The more hydrophilic 15-residue signal peptide of the mannitol permease (mtl15) also incorporated into monolayers and detergent micelles (although to a lesser extent) with induction of secondary structure. Based on these results and a parallel with mitochondrial targeting in eucaryotes, we suggest that the induction of N-terminal amphiphilic structures and their association with a hydrophobic-hydrophilic interface are important for envelope targeting and the initiation of the membrane insertion of bacterial phosphoenol-pyruvate-dependent phosphotransferase system permeases.     

Pharmacological profile of a model for central serotonin receptor activation

The head shake response in rats after systemic administration of the serotonin (5HT) precursor 5-hydroxytryptophan (5HTP) was pharmacologically characterized and shown to be a useful animal model to quantify brain 5HT receptor activation. The behavior occurred in a dose-dependent manner after injection of 5HTP and the 5HT agonist quipazine. Head shakes were also observed after injection of L-tryptophan, 5-methoxydimethyltryptamine and fenfluramine. The 5HT antagonists cyproheptadine and metergoline were potent blockers of the response. Xylamidine, a peripheral 5HT antagonist, had no effect on head shaking. Inhibition of 5HT uptake with fluoxetine potentiated the head shake response after 5HTP. Manipulation of central cholinergic or GABAergic mechanisms did not alter 5HTP-induced shakes. Alpha-noradrenergic receptor blockade had no significant effect on head shakes. However, desmethylimipramine was equipotent with methysergide as an antagonist of the behavior. Beta-noradrenergic receptor blockade had no specific effect on 5HTP head shakes. Concomitant dopamine receptor activation with SK&;F 38393 did not affect head shakes but the neuroleptics chlorpromazine and pimozide reduced the number of head shakes after 5HTP. The H₁ receptor antagonist pyrilamine had no effect on head shakes. It is concluded that 5HTP-induced head shakes in rats is a quantitative model of brain 5HT receptor activation which is particularly sensitive to 5HT antagonists.     

Lipophilicity Determination of Highly Lipophilic Compounds by Liquid Chromatography

Different experimental strategies using short columns in both conventional liquid chromatography (HPLC) and ultra‐high pressure liquid chromatography (UHPLC) were evaluated to allow, for the first time with these techniques, the lipophilicity determination of compounds with log P>5. Various organic modifiers, stationary phases, and elution modes were tested on 14 rigid compounds with a CLogP between 5 and 8, and 38 compounds with log P_oct from 0 to 5. The best results in HPLC were obtained with the 20‐mm Discovery ^® RP Amide C16 stationary phase in isocratic mode using MeOH as organic modifier. To improve analysis time, the UHPLC approach was then evaluated. Consequently, a generic method was developed with a 30‐mm Acquity BEH Shield RP18 column in gradient mode using MeOH as organic modifier, allowing a fourfold gain of time compared to the HPLC method, for the highly lipophilic compounds tested. Finally, the most rapid and accurate results were obtained with a 10‐mm Hypersil^TM GOLD Javelin HTS stationary phase in UHPLC, enabling an eightfold gain of time compared to the HPLC method.     

Alteration of bacterial surface electrostatic potential and pH upon adhesion to a solid surface and impacts to cellular bioenergetics

In our previous study [Hong Y, Brown DG (2009) Appl Environ Microbiol 75(8):2346–2353], the adenosine triphosphate (ATP) level of adhered bacteria was observed to be 2–5 times higher than that of planktonic bacteria. Consequently, the proton motive force (Δp) of adhered bacteria was approximately 15% greater than that of planktonic bacteria. It was hypothesized that the cell surface pH changes upon adhesion due to the charge‐regulated nature of the bacterial cell surface and that this change in surface pH can propagate to the cytoplasmic membrane and alter Δp. In the current study, we developed and applied a charge regulation model to bacterial adhesion and demonstrated that the charge nature of the adhering surface can have a significant effect on the cell surface pH and ultimately the affect the ATP levels of adhered bacteria. The results indicated that the negatively charged glass surface can result in a two‐unit drop in cell surface pH, whereas adhesion to a positively charged amine surface can result in a two‐unit rise in pH. The working hypothesis indicates that the negatively charged surface should enhance Δp and increase cellular ATP, while the positively charged surface should decrease Δp and decrease ATP, and these results of the hypothesis are directly supported by prior experimental results with both negatively and positively charged surfaces. Overall, these results suggest that the nature of charge on the solid surface can have an impact on the proton motive force and cellular ATP levels. Biotechnol. Bioeng. 2010;105: 965–972. © 2009 Wiley Periodicals, Inc.     

Octanol–water partition of nonzwitterionic peptides: Predictive power of a molecular size-based model

A remarkably simple, molecular size-based model developed to predict octanol–water partition coefficients for organic compounds is tested on a set of 188 neutral peptides with available experimental partition data. Despite using only two parameters, it gives a promising correlation (r² = 0.914; σ = 0.455, F = 1978.0), and predictions are in a realistic range even for larger peptides (cyclosporin, melanotan, sandostatin) where common, overparametrized fragment methods become quite unreliable. Ion-pair partitioning and the extraction constant formalism is briefly reviewed to describe the sigmoidal lipophilicity profile of ionizable, nonzwitterionic peptides. It seems possible to extend the present model to estimate apparent partition coefficients measured around neutral pH and physiological conditions for monoionic peptides; however, as no standard conditions are yet defined and only relatively small number of experimental data are available, the situation here is more complex. Proteins 30:86–99, 1998. © 1998 Wiley-Liss, Inc.     

Effect of reserpine on 5-hydroxytryptophan (5HTP)-immunoreactive neurons in the rat brain

By immunohistochemistry of rat brain in conjunction with a specific antibody against 5-hydroxytryptophan (5HTP), we examined immunoreactivity to 5HTP in neurons, from which 5-hydroxytryptamine (5HT; serotonin) was depleted by reserpine treatment. The distribution patterns of 5HTP-positive neurons overlapped with those of 5HT neurons. Treatment with reserpine (5 mg/kg, 90 min before death) caused a complete suppression of 5HT-positive staining, but 5HTP-immunostaining remained in perikarya of the nuclei raphe dorsalis, centralis superior and obscurus. Treatment with reserpine (25 mg/kg, 90 min before death) suppressed the 5HTP-immunoreaction in certain perikarya (e.g. of the nucleus raphe dorsalis) and fibres; however, 5HTP-immunostaining remained in perikarya of the nuclei centralis superior and raphe obscurus. This suggests that these neurons synthesize more 5HTP by a process which appears to be stimulated by reserpine.     

HPLC-based lipophilicity of pyrrolyl-acetic acid ARIs: Relationships with biological activity

Reversed phase HPLC was used to assess the lipophilicity of a series pyrrolyl-acetic acid derivatives with aldose reductase inhibitory activity. The pH conditions were adjusted at 3.0 to investigate the behavior of the neutral species and at pH 7.4, at which the ionized form predominates, using phosphate and MOPS buffer. Retention was monitored in absence and in presence of different amounts of n-octanol in the mobile phase in order to explore the chromatographic conditions which best reproduce the octanol–water partition or distribution coefficients. The effect of n-octanol in retention was systematically studied and its role in lipophilicity assessment was evaluated. Nevertheless rather moderate regression equations were obtained, which deviated significantly from the ideal 1:1 correlation. No significant effect of buffer was observed. The appropriateness of retention factors to be used in correlation with aldose reductase inhibitory activity was further evaluated and compared to the efficiency of the corresponding octanol–water log P values.     

Optimization of reversed-phase microcapillary liquid chromatography for quantitative proteomics

A specific capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) method for the determination of serotonin (5HT) and its precursors tryptophan (Trp) and 5-hydroxytryptophan (5HTP) in human platelet rich plasma is described. The analytes were removed from the plasma samples and preconcentrated by solid phase extraction (SPE) on mixed mode cation-exchange sorbents. The SPE recoveries were 71.6 +/- 3.1 for 5HT, 91.0 +/- 2.8 for Trp, and 95.3 +/- 5.9% for 5HTP. Deuterated analogues of 5HT and Trp were used as internal standards for quantitation purposes. Submicromolar detection limits were obtained for standard mixtures of all compounds and their deuterated isotopes, except 5HTP, which had detection limits in the low micromolar range. The potential usefulness of this method in the clinical setting was demonstrated by analyzing plasma extracts from healthy volunteers as well as from pathological samples. While 5HTP was not present in any of the analyzed samples, the levels of 5HT and Trp in both normal and pathological plasma were determined.     

Neural control of olfaction and tentacle movements by serotonin and dopamine in terrestrial snail

We investigated the role of serotonin (5HT) and dopamine (DA) in the regulation of olfactory system function and odor-evoked tentacle movements in the snail Helix. Preparations of the posterior tentacle (including sensory pad, tentacular ganglion and olfactory nerve) or central ganglia with attached posterior tentacles were exposed to cineole odorant and the evoked responses were affected by prior application of 5HT or DA or their precursors 5-hydroxytryptophan (5HTP) and l-DOPA, respectively. 5HT applications decreased cineole-evoked responses recorded in the olfactory nerve and hyperpolarized the identified tentacle retractor muscle motoneuron MtC3, while DA applications led to the opposite changes. 5HTP and l-DOPA modified MtC3 activity comparable to 5HT and DA action. DA was also found to decrease the amplitude of spontaneous local field potential oscillations in the procerebrum, a central olfactory structure. In vivo studies demonstrated that injection of 5HTP in freely moving snails reduced the tentacle withdrawal response to aversive ethyl acetate odorant, whereas the injection of l-DOPA increased responses to “neutral” cineole and aversive ethyl acetate odorants. Our data suggest that 5HT and DA affect the peripheral (sensory epithelium and tentacular ganglion), the central (procerebrum), and the single motor neuron (withdrawal motoneuron MtC3) level of the snail’s nervous system.     

Role of stationary phase and eluent composition on the determination of log P values of N-hydroxyethylamide of aryloxyalkylen and pyridine carboxylic acids by reversed-phase high-performance liquid chromatography

The partition coefficients, P, between n-octanol and water of a number of growth stimulating substances, N-hydroxyethylamide of aryloxyalkylen- and pyridine carboxylic acids were obtained from Pomona College (C log P), and Rekker's (log P_Rekker) revised fragmental constant system was used to calculate log P data sets. Both of these data sets were correlated with two different substance lipophilicity parameters, log k_w and ₀. Log k_w was obtained by extrapolation of log retention factor (k) to 0% organic modifier measured in reversed-phase liquid chromatography (RPLC) systems. ₀ values were obtained from the slopes and intercepts of these relationships. The RPLC experiments were performed on four commercially available reversed-phase columns. Binary mixtures of methanol–water, methanol–phosphate buffer (pH 7.0), methanol–tricine buffer (pH 7.0) and acetonitrile–water were used as mobile phases for the determination of log k_w values. For the methanolic eluents linear regression provided satisfactory correlations (r>0.99) for the relationships log k vs. organic modifier content in the eluent, while for the acetonitrile-containing eluents a second-degree polynominal regression was necessary. For all four RPLC columns, by linear regression satisfactory correlations (r>0.99) were obtained between log k_w and log P data using methanolic eluents. In such eluents ₀ values were shown to be the second-best lipophilicity parameters. For acetonitrile-containing eluents the use of second-degree polynominal regression was necessary and, in contrast to methanol, significant influence of the applied column on regression results was observed. For acetonitrile-containing eluents the ₀-index does not provide satisfactory results for our substances. No difference in regression results between the use of buffered and non-buffered eluents was observed.     

The Plasmodium falciparum exportome contains non‐canonical PEXEL/HT proteins

The pathogenicity of Plasmodium falciparum is partly due to parasite‐induced host cell modifications. These modifications are facilitated by exported P. falciparum proteins, collectively referred to as the exportome. Export of several hundred proteins is mediated by the PEXEL/HT, a protease cleavage site. The PEXEL/HT is usually comprised of five amino acids, of which R at position 1, L at position 3 and E, D or Q at position 5 are conserved and important for export. Non‐canonical PEXEL/HTs with K or H at position 1 and/or I at position 3 are presently considered non‐functional. Here, we show that non‐canonical PEXEL/HT proteins are overrepresented in P. falciparum and other Plasmodium species. Furthermore, we show that non‐canonical PEXEL/HTs can be cleaved and can promote export in both a REX3 and a GBP reporter, but not in a KAHRP reporter, indicating that non‐canonical PEXEL/HTs are functional in concert with a supportive sequence environment. We then selected P. falciparum proteins with a non‐canonical PEXEL/HT and show that some of these proteins are exported and that their export depends on non‐canonical PEXEL/HTs. We conclude that PEXEL/HT plasticity is higher than appreciated and that non‐canonical PEXEL/HT proteins cannot categorically be excluded from Plasmodium exportome predictions.